Protein folding studies are directed towards the goal of achieving the capability of designing novel polypeptide sequences which will fold into desired shapes with medically useful activities. A potentially useful class of synthetic proteins would be small inhibitors of enzymes. Many natural inhibitors consist of globules with a protruding amino acid which resembles the enzyme's substrate, such that the inhibitor competes for enzyme active sites in binding reactions. Enzyme inhibitors regulate many physiological processes such as complement fixation, blood-clotting, ovum fertilization, and cell-cell surface interactions. The objective of the proposed research is to develop strategies for directing formation of specific disulfides in a solution containing many different cysteines. The proposed research will study disulfide pairing between cysteine-containing fragements obtained from inexpensive commercially available proteins. Success would suggest synthetic containing portions of the same fragments in a single chain, yielding the same specific pairing with a resultant specific topology. Different sequences could be designed for producing a collection of novel mainframes. These would serve as the basis for subsequent inhibitor production by addition of protruding amino acids at different locations on the mainframes. This proposal will test the hypothesis that the formation of specific bridges can be controlled by choosing appropriate positions for the cysteines with appropriate positive, negative, or aromatic residues. Experiments will measure quantitative effects of such factors on the microscopic rate constants in disulfide exchange reactions. These rates will be used to suggest peptide mixtures where particular disulfides are predicted to predominate at equilibrium. The predictions will be evaluated experimentally by establishing disulfide interchange equilibria between protein fragments, separating the products and determining their relative concentrations.